EP1445260A1 - Méthode de la immobilisation des composés chimiques à des phases solides - Google Patents

Méthode de la immobilisation des composés chimiques à des phases solides Download PDF

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Publication number
EP1445260A1
EP1445260A1 EP03002404A EP03002404A EP1445260A1 EP 1445260 A1 EP1445260 A1 EP 1445260A1 EP 03002404 A EP03002404 A EP 03002404A EP 03002404 A EP03002404 A EP 03002404A EP 1445260 A1 EP1445260 A1 EP 1445260A1
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EP
European Patent Office
Prior art keywords
immobilized
compound
contacting
coupling reagent
base
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP03002404A
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German (de)
English (en)
Inventor
Karsten Dr. Schnatbaum
Ulf Dr. Reimer
Dirk Dr. Scharn
Mike Dr. Schutkowski
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Jerini AG
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Jerini AG
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Priority to EP03002404A priority Critical patent/EP1445260A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support

Definitions

  • the present invention relates to methods for forming a chemical bond between a first functional group immobilized on a surface and a second functional group that is part of a compound to be immobilized.
  • Biological test reactions such as enzymatic conversion or antibody binding are very good sensitive, so that small amounts ( ⁇ g to ng) of peptide substrates are required.
  • the peptide substrate is immobilized on a solid support material and easily removed from the reaction solution can be.
  • the reaction with the peptide can then either in the remaining Reaction solution or by subsequent analysis of the carrier-bound material be measured quantitatively.
  • the unactivated form of the compound to be coupled is converted into the corresponding activated form by a suitable reagent in a solution and used in situ for the desired coupling reaction without isolation of the active derivative.
  • reagents that have been used for the in situ synthesis of activated derivatives are TBTU with the base DIPEA [R. Volkmer-Engert et al., Tetrahedron Lett. 1997, 38 , 1029-1032], PyBOP with the base DIPEA [A. Kramer et al., Methods: A Companion to Methods in Enzymology, 1994, 6 , 388-395], DIC with the base NMI [U. Hoffmüller et al., Angew . Chem .
  • the present invention was therefore based on the object of a method for training a chemical bond, in particular a process for the production of polypeptides, To provide which has the disadvantages of the methods known in the prior art overcomes. It was a particular object of the present invention to provide a method to provide a fully automated synthesis of peptides on a particular planar surface allowed.
  • the coupling reagent and / or the Compound at least one additive is added.
  • connection and / or the Coupling reagent the base is added.
  • the base is represented by the immobilized functional group.
  • the method according to the invention further includes the step:
  • this step being separated, in particular separate from the steps of contacting the surface with the Coupling reagent and contacting the surface with the one to be immobilized Connection is established.
  • the The surface is contacted at a defined point on the surface.
  • step a) and step b) at the essentially same place he follows.
  • the Contacting the surface with the base takes place in essentially the same place as contacting the surface with the coupling reagent and the one to be immobilized Connection.
  • step a) when repeating step a) the compound to be immobilized the same or different compound to be immobilized is compared to that of the previous step.
  • the Coupling reagent and / or at least one additive is / is added to the compound.
  • the step includes:
  • this step being separated, in particular separate from the step of contacting the surface with the Coupling reagent and / or the step of contacting the surface with the Coupling reagent and the compound to be immobilized.
  • the coupling reagent, the additive, the base and / or the Connection is in a solution.
  • the bond formed is a bond selected from the group consisting of Carboxylic acid amide bond, carboxylic acid ester bond, carboxylic acid anhydride bond, Sulfonic acid amide bond, phosphoric acid ester bond, phosphonic acid ester bond, Phosphorous acid ester bond and phosphoric acid amide bond.
  • the Coupling reagent is selected from the group consisting of carbodiimides, aminium and Phosphonium salts comprises, is preferably selected from the group consisting of HBTU, Includes TBTU and PyBOP.
  • the Coupling reagent is present in a solvent and the solvent is preferably is selected from the group consisting of N, N-dimethylformamide, 1-methyl-2-pyrrolidinone, N, N-dimethylacetamide, Dimethyl sulfoxide, tetrahydrofuran, dioxane, acetonitrile, methanol, Ethyl acetate, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol, 1,2-dibromoethane, Includes dichloromethane, diethyl ether and acetone.
  • the solvent is preferably is selected from the group consisting of N, N-dimethylformamide, 1-methyl-2-pyrrolidinone, N, N-dimethylacetamide, Dimethyl sulfoxide, tetrahydrofuran, dioxane, acetonitrile, methanol, Ethyl acetate, 2,2,2-trifluoroethanol
  • the additive is selected from the group comprising benzotriazoles, benzotriazines, phenols and organic and inorganic salts, preferably the additive is selected from the group consisting of HOBt, HOAt, HOOBt, HOPfp, Includes NaClO 4 and KSCN.
  • the Aggregate is present in a solvent and the solvent is preferably is selected from the group consisting of N, N-dimethylformamide, 1-methyl-2-pyrrolidinone, N, N-dimethylacetamide, Dimethyl sulfoxide, tetrahydrofuran, dioxane, acetonitrile, methanol, Ethyl acetate, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol, 1,2-dibromoethane, Includes dichloromethane, diethyl ether and acetone.
  • the solvent is preferably is selected from the group consisting of N, N-dimethylformamide, 1-methyl-2-pyrrolidinone, N, N-dimethylacetamide, Dimethyl sulfoxide, tetrahydrofuran, dioxane, acetonitrile, methanol, Ethyl acetate, 2,2,2-trifluoroethanol, 1,1,
  • the Compound to be immobilized is selected from the group consisting of carboxylic acids, Carboxylic acid esters, sulfonic acids, phosphoric acid esters, phosphonic acid esters, Phosphoric acid esters, phosphoramidites, primary amines, secondary amines and alcohols comprises, and the compound is preferably selected from the group consisting of Carboxylic acids, primary amines and secondary amines.
  • the Compound is in a solvent and the solvent is preferably is selected from the group consisting of N, N-dimethylformamide, 1-methyl-2-pyrrolidinone, N, N-dimethylacetamide, Dimethyl sulfoxide, tetrahydrofuran, dioxane, acetonitrile, methanol, Ethyl acetate, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol, 1,2-dibromoethane, Includes dichloromethane, diethyl ether and acetone.
  • the solvent is preferably is selected from the group consisting of N, N-dimethylformamide, 1-methyl-2-pyrrolidinone, N, N-dimethylacetamide, Dimethyl sulfoxide, tetrahydrofuran, dioxane, acetonitrile, methanol, Ethyl acetate, 2,2,2-trifluoroethanol, 1,1,1,3,
  • the Base is selected from the group consisting of tertiary amines, pyridines, imidazoles and organic Salts, preferably the base is selected from the group consisting of DIPEA, Includes collidine, DMAP, NMI, NMM, pyridine and KOBt.
  • the Base is present in a solvent
  • the solvent preferably being selected is from the group consisting of N, N-dimethylformamide, 1-methyl-2-pyrrolidinone, N, N-dimethylacetamide, Dimethyl sulfoxide, tetrahydrofuran, dioxane, acetonitrile, methanol, Ethyl acetate, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol, 1,2-dibromoethane, Includes dichloromethane, diethyl ether and acetone.
  • the immobilized functional group is selected from the group consisting of carboxylic acids, Carboxylic acid esters, sulfonic acid, phosphoric acid esters, phosphonic acid esters, Phosphoric acid esters, phosphoramidites, primary amines, secondary amines and alcohols comprises and which is preferably selected from the immobilized functional group the group comprising carboxylic acids, primary amines and secondary amines.
  • contact is made by contacting a capillary.
  • that contact is made by stamping.
  • the object is achieved in a seventh aspect by a surface with a chemical bond between a first functional group and a second functional group, the second functional group being part of a surface immobilized compound is producible by a method according to the present Invention.
  • the present invention is based on the surprising finding that it is due to the specific sequence and temporal separation of the steps of contacting the surface with the coupling reagent and contacting the surface with the compound to be immobilized or that the reactive species of the compound to be immobilized only on the Surface is formed, it is possible to avoid the disadvantages known in the prior art and described above of a method for forming a chemical bond, in which the coupling of typically derivatized amino acids takes place, the amino acids being present as activated derivatized amino acids before the coupling.
  • One such method is the SPOT method known in the prior art.
  • the stability problems observed when using activated amino acid derivatives in particular in the event that the activated derivatives are kept in the presence of bases avoided.
  • Prolonged standing of amino acids which were preactivated with the coupling reagent HATU and the base collidine led to increased racemization of the amino acid [LA Carpino et al., Tetrahedron 1999, 55 , 6813-6830] and to rapid decomposition.
  • solutions of pure coupling reagents or solutions of coupling reagents with bases are often stable for hours [F. Albericio et al., J. Org. Chem. 1998, 63 , 9678-9683] [F. Albericio et al., Tetrahedron Lett. 1997, 38 , 4853-4856].
  • the methods according to the invention allow, insofar as it is too immobilizing compound is an amino acid that acts as the starting materials amino acids used for the synthesis may be in a form which allow long-term storage, so that in automated embodiments of the inventive method a time and material intensive pre-activation before each Clutch cycle is not required.
  • the compound to be immobilized is an amino acid or a peptide with a carboxyl and an amino group ensures that the Number of undesirable side reactions due to the simultaneous presence of both Carboxyl and amino groups is reduced.
  • Solid Phase peptide synthesis Solid Phase Peptide Synthesis, SPPS
  • SPPS Solid Phase Peptide Synthesis
  • the second functional Group is part of a compound to be immobilized, the surface being provided and with the compound to be immobilized thereon and a coupling reagent and optionally an aggregate and also optionally contacted with a base and is implemented, it is provided that the steps of contacting the surface with the Coupling reagent and contacting the surface with the one to be immobilized Connection made separately. It is particularly provided that the implementation of the The above two steps are carried out separately from one another. It is in the Framework of the present invention that first the coupling reagent with the surface is contacted and then the surface with the compound to be immobilized. Alternatively, it is possible that the surface is first immobilized Connection and then contacted with the coupling reagent.
  • the addition or the presence of a Base the formation of the bond between the immobilized functional group and the second functional group positively.
  • the presence of the base can either by adding an appropriate base, preferably in a suitable one Solvents, take place, or by the individual, in reaction with each other bringing components or agents, d. H. connection to be immobilized, Coupling reagent and optional aggregate, by one of them alone or in Combination with one or more of those to be reacted with one another Components are shown or trained.
  • the coupling reagent first the surface is given by contacting and then a mixture of immobilizing compound and base.
  • the Mixture of compound to be immobilized and base and then the coupling reagent is given up on the surface.
  • the addition takes place of the individual components, d. H. the compound to be immobilized, the Coupling reagent and the base, one after the other in time, basically all Permutations of the order in which the components are applied to the surface possible are.
  • the compound to be immobilized, the coupling reagent and the base are preferably applied to the surface in separate work steps.
  • the surface can first with the coupling reagent, followed by the base and then contacted with the compound to be immobilized.
  • the surface can be contacted with the coupling reagent first, followed by Contacting the surface with the compound to be immobilized and finally Contact the surface with the base.
  • the surface is first contacted with the base, then with the immobilizing compound and finally with the coupling reagent.
  • the surface first with the base, then with the Coupling reagent and finally contacted with the compound to be immobilized becomes.
  • the surface is first contacted with the compound to be immobilized, then with the Coupling reagent and finally with the base.
  • the surface first with the one to be immobilized Compound, then contacted with the base and finally with the coupling reagent becomes.
  • one or more of the above takes place after each order mentioned agents a drying step, but at least the individual agents or mixtures, as stated above, contacted separately with the surface. It is it within the scope of the present invention that contacting the surface with the different agents in essentially the same place. It is also in the Within the scope of the present invention that the contacting of individual agents on a larger part of the surface than contacting one or more of the surfaces other agents.
  • Agents are generally used here in particular immobilizing compound, the coupling reagent, the base and the additive designated. It is within the scope of the present invention that the aggregate can basically be added at any time in the reaction process.
  • the contacting of the Surface with the coupling reagent separated in time from contacting the Surface with the compound to be immobilized and the base.
  • the two steps are to be carried out preferably in this order.
  • the time interval between The individual contacting steps can be as little as 0.01 seconds and as much as 5 hours.
  • Contacting the surface with the one to be immobilized Connection and the base can be designed so that the immobilized Compound and the base are present together in a solution or solvent and the both containing solution or the solvent containing both by contacting the solution or the solvent are deposited with the surface on the surface.
  • the immobilized Contact is made with the surface and the base with the Surface is contacted.
  • the contacting of the surface with the compound to be immobilized and the base take place very quickly in succession, whereby it is both possible to first deposit the base and then the one to be immobilized Compound as well as first the compound to be immobilized and then the base.
  • the Temporal framework conditions are preferably such that the preferably in Solution settled compound to be immobilized has not yet dried out Time when the base is added. The same applies to the case that the base is discontinued first. Typical time intervals depend on the respective Experimental conditions, such as humidity and temperature. at Room temperature and normal humidity is the time interval for this form performing the contacting of the surface with the compound to be immobilized and the base as little as 0.01 sec and as much as 1 hour.
  • connection to be immobilized is only activated when it is on the Surface is present or is or is deposited on this.
  • the term surface preferably denotes that Surface of a carrier material.
  • the training is a chemical bond, preferably a peptide bond.
  • the immobilized functional group of one already immobilized amino acid can preferably either be an amino group or a carboxyl group.
  • second functional group is a carboxyl group or amino group, which is further from the immobilizing compound, preferably also one in the present case Amino acid originates from the first already immobilized amino acid by training bound to a peptide bond and thus immobilized.
  • linker between the surface and the synthesized peptide, which can be attached to the surface both with the methods according to the invention and with conventional methods.
  • linkers are known to the person skilled in the art [F. Guillier et al., Chem . Rev 2000, 100 , 2091-2157].
  • the peptide can also be synthesized directly on the surface without a linker if the surface is suitably functionalized, such as, for example, with OH groups on which the peptide is immobilized via an ester bond.
  • each Contacting step takes place at a defined point on the surface. It is preferred such a defined location on the surface is a partial area of the surface. It's like that in one Embodiment of the inventive method provided that the contact on on a surface and the location has been previously determined or determined. This is especially necessary if there are several spots on the surface to whom a connection is immobilized or immobilized and to whom the method according to the invention is preferably carried out in parallel or sequentially becomes.
  • a defined site is also referred to herein as a reaction site.
  • polymers at defined points in particular peptides, which are also generally referred to herein as Polypeptides are referred to, or have polynucleotides with a defined sequence.
  • the coupling reagent either selectively at the reaction site, at the reaction site and a surrounding area Area, or is applied overall on the surface.
  • the compound to be immobilized and the base on either this reaction site, or in an area resulting from the coupling reagent Contacting the surface is equipped with the coupling reagent, discontinued or applied.
  • a larger area of the surface than the reaction site with coupling reagent is equipped and in this area, preferably again at a specific point thereof, the compound to be immobilized and the base by contacting the surface to be provided.
  • the surface with the Coupling reagent and the compound to be immobilized is contacted while doing so these are placed on it.
  • the coupling reagent and the connection to be immobilized at the defined point on the surface be contacted or dropped off there.
  • this is done Contact the surface with the base. It can be provided that contacting the surface with the base corresponds to the point at which according to step a) Coupling reagent and the compound to be immobilized contact the surface or were placed on it.
  • the area of the surface that is contacted with the base over the area or the Place the surface with the coupling reagent and the one to be immobilized Connection is contacted extends. It is also within the scope of the invention Process that the contact of the surface with the base is designed so that the entire surface is contacted with the base.
  • the second functional Group is part of a compound to be immobilized, each of the steps as described in the In connection with the above methods have been described, and in which a Agent or a mixture of several agents is contacted with the surface first at each of the plurality of locations before the further step at the Most of the positions are carried out.
  • the surface is contacted with the coupling reagent and the connection to be immobilized basically in the same way as for the Process according to the invention for forming a chemical bond described.
  • the step a) is repeated at least once. The repetition is preferably carried out at a different location than in each case previous step. However, the repetition can also be in the same place or Reaction point.
  • step c) Contacting the surface with the base according to step c) can take place in such a way that the individual points or reaction points on the surface at which contact with the coupling reagent and the compound to be immobilized is done, can be contacted individually with the base.
  • the individual points or reaction points on the surface at which contact with the coupling reagent and the compound to be immobilized is done can be contacted individually with the base.
  • This method differs from the previously described invention Process for the parallel formation of a chemical bond in that for each single reaction point after contacting the surface or the reaction point on the coupling reagent and the compound to be immobilized The surface is contacted with the base. This sequence of steps is then repeated for each of the defined positions. It is within the scope of the present Invention that step a) is repeated several times at the same point before step b) is carried out. It is also within the scope of the present invention that the step a) is carried out at several different defined locations before contacting the surface is done with the base. It is particularly preferred that contacting the Base in the same or approximately the same defined position or the same defined places as in step a). This sequence of repeating the step a) and step b) can then be repeated at least once. With each of the Methods according to the invention for the parallel formation of a chemical bond apply with regard to the design of the individual process steps and the selection of functional groups basically those in connection with the invention A method of forming a chemical bond features disclosed therein and Embodiments.
  • the compound to be immobilized is compared to that connection which is in the the previous step is used at this or another reaction point.
  • Processes in preferred embodiments around processes for the synthesis of polymers, especially of polypeptides, the compounds to be immobilized then Are amino acids.
  • Coupling reagents are used in the process according to the invention used.
  • Coupling reagents are those substances which the reactions disclosed herein, in particular training the various Binding types, enable.
  • Coupling reagents which bind a Lead amide bond as shown for example in Fig. 3.
  • the various coupling reagents that can be used according to the invention can be assigned to specific subgroups.
  • An important group is that of carbodiimides (X3) , which react to form ureas in peptide couplings with H 2 O uptake.
  • Formula X3 shows such a carbodiimide.
  • R 1 , R 2 alkyl, cycloalkyl, substituted alkyl, substituted cycloalkyl.
  • alkyl refers to any straight or branched chain alkane with a chain length of C1 to C12. Chain lengths from C1 to C6 are preferred.
  • the alkyl radical is a cycloalkyl.
  • the cycloalkyl preferably consists of three to 12 carbon atoms (C3 to C12), preferably from five to seven carbon atoms (C5 to C7).
  • C1 to C12 denotes any number of carbon atoms from 1 to 12, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The same applies mutatis mutandis to the corresponding others Speed ranges.
  • carbodiimides which can be used according to the invention are:
  • N, N'-dicyclohexylcarbodiimide (DCC, DCCI), 1-ethyl-3- (3'dimethylaminopropyl) carbodiimide ⁇ HCl (EDC ⁇ HCl, WSC ⁇ HCl) and diisopropylcarbodiimide ( DIC ).
  • aminium salts (X1) and phosphonium salts (X2) are preferably used.
  • radicals R2 and R4 and / or R3 and R5 are capable of forming a ring and so far heteroaromatic ring is formed. It is also possible that R2 and R3 and / or R4 and R5 can form a ring, which also makes a heteroaromatic ring is trained.
  • the HBTU example explains very positive results.
  • the generally accepted mechanism of peptide bond formation is shown in FIG. 4. With extremely short preactivation times, as in the case of the present invention, a larger part of the peptide bond can be formed via the intermediate X6 of FIG. 4.
  • X6 is more reactive than X7 (OAt ester) [LA Carpino et al., Angew. Chem. 2002, 114 , 458-461].
  • This difference in the active species compared to the previously known methods can have a number of positive effects on the coupling process: racemization can be less starting from X6 than starting from X7 in FIG. 4, the reaction can proceed faster and the yields can be increased.
  • HBTU does not activate a carboxylic acid, as is clear from the NMR spectra in FIG. 7. That explains the high stability of Solutions made from HBTU and Fmoc-AS-OH in DMF that work even when left to stand for one day the air still has an average of about 70% of its original activity (see Fig. 8).
  • Phosphonium reagents react in a very similar mechanism to aminium reagents, the mechanism being described in FIG. 5.
  • aminium and phosphonium coupling reagents as described in the Processes according to the invention can be used:
  • aminium and phosphonium coupling reagents those reagents which are based on hydroxybenzotriazoles such as HOBt (X6) and HOAt ( X7 ) are particularly preferably used in the processes according to the invention.
  • HBTU (X3), TBTU (X4) and PyBOP (X5) are very particularly preferred in the context of the present invention .
  • Two chemical structures have been described for HBTU in the past. Structure B was initially proposed [V. Dourtoglou et al., Synthesis 1984, 572], later structure A [I. Abdelmoty et al., Lett . Pept. Sci . 1994, 1 , 52], and it has recently been known that both structures exist [LA Carpino et al., Angew. Chem. 2002, 114 , 458-461].
  • the name HBTU expressly includes both possible isomers. The same applies to connections that are structurally related to the HBTU.
  • the coupling process is often positively influenced by the addition of bases.
  • Bases as they can be used according to the method according to the invention, are also generally those according to the definition of Bronsted compounds which can act as proton acceptors [AF Hollemann, N. Wiberg, Textbook of Inorganic Chemistry , Walter de Gruyter, New York , 1985 , p.236]. This property is particularly advantageous or advantageous in the formation of amide bonds, since the desired coupling step is often initiated by the abstraction of a proton from the carboxyl group of the carboxylic acid (cf. FIG. 3).
  • additives can be used, in particular additives in addition to the coupling reagent or to the immobilizing compound.
  • Aggregates as used herein are compounds that are suitable for the desired ones To influence the coupling process positively. Suitable additives can be obtained from the Experts in the field also in the context of routine tests with regard to their suitability for Use in one of the methods according to the invention can be determined or determined.
  • HOBt pentachlorophenol
  • 2,4-dinitrophenol are suitable for reducing the undesired base-catalyzed formation of aminosuccinyl peptides from aspartyl peptides in peptide couplings.
  • the underlying mechanism is not known here, but it is assumed that the weakly acidic compounds buffer the base used [J. Martinez et al., Int. J. Pept. Prot. Res. 1978, 277].
  • Additives that can be used by way of example in the context of the present invention include 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), 1-hydroxybenzotriazole ⁇ H 2 O (HOBt ⁇ H 2 O), 6 -Chlor-1-hydroxybenzotriazole (6-Cl-HOBt), ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate (HOCt), N-hydroxy-5-norbornenendo-2,3-dicarboximide (HONB), N-hydroxysuccinimide (NHS, HOSu), 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt, HOOBt), pentafluorophenol (HOPfp), lithium chloride (LiCl), Sodium perchlorate (NaClO4), potassium rhodanide (KSCN), copper (II) chloride (CuCl
  • the base KOBt [KM Sivanandaiah et al., Int. J. Pept. Prot. Res. 1994, 44 , 24-30] can also be regarded as an additive, since the additive HOBt is formed in couplings with KOBt, in which acids are released.
  • the compounds listed as additives can perform the function of a base and thus be used as a base in the process according to the invention and bases as additives, at least to a certain extent.
  • a drying step is provided within the scope of the method according to the invention is generally favorable when drying the treated surface or a part thereof, dry between two or more of the steps. Preferably there is a Drying at least before the last step in time. This has the advantage that after the last step, followed by the activation and coupling of the immobilizing connection to the immobilized group takes place, the highest possible Concentration of activated derivative can be achieved. Higher concentrations Reaction partners usually lead to higher reaction rates. Becomes on the other hand, if not dried, the solvent used in the first steps leads to smaller concentrations of the active derivative ultimately required.
  • Preferred embodiments of the method according to the invention provide that the Drying between application of coupling reagent and a compound-base mixture and / or between the application of a compound-coupling reagent mixture and a base is done.
  • a washing step is also provided in which the surface is freed of agents or their extent on the surface is reduced becomes.
  • a solvent is used.
  • a washing step is preferably carried out after completion of the desired chemical Reaction before performing a new reaction. This prevents during the second reaction disrupt reagents from the first reaction. For practical execution the surface can be showered with solvent in a bowl and shaken become.
  • the successive washing of the surface with different ones is preferred Solvents, washing after the completion of the reaction after each Reaction step takes place.
  • the different washing steps are different Solvent used.
  • solvents Different polarity has advantages here because it offers an even wider bandwidth different non-covalently bound substances on the surface can be removed than would be the case with a single solvent.
  • An example is the sequential washing with DMF, MeOH and DCM after each step.
  • the various compounds i. H. the connection to be coupled or the connection to be immobilized, the Coupling reagent, the additive and / or the base present in solvents used. It is also within the scope of the invention that two or more of the the aforementioned compounds simultaneously in a solvent or in the same Solvents are included.
  • solvents in which the compound to be immobilized or coupled, the coupling reagent or the additive can be present N, N-dimethylformamide (DMF), 1-methyl-2-pyrrolidinone (NMP), N, N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), dioxane, acetonitrile (MeCN, ACN), methanol ( MeOH), ethyl acetate (EE), 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (hexafluoroisopropanol, HFIP), 1,2-dibromoethane, dichloromethane ( DCM), diethyl ether, acetone, 1,2-dimethoxyethane (DME), bis (2-methoxyethyl) ether (diglyme), 1,3-dimethyl-2-imidazolidinone, 1,
  • Solvent For general selection of the appropriate solvent, the person skilled in the art Different criteria. An important criterion of which to use Solvent is its property, the respective substance or substances to solve in high concentrations. This has the advantages of high concentrations fast response speeds can be achieved and the amount of expensive and environmentally harmful solvents can be reduced. But this also has the effect that the solvent and the compound (s) contained therein at higher speed can be distributed by a pipetting device, which is particularly true in parallel synthesis very many different connections and in the context of an automated The inventive method is not insignificant.
  • Volatility is another important criterion for choosing a suitable solvent the same and the time at which it is used. Following the first Steps of the process in which there is little or no training If the desired chemical bond occurs, drying the surface is beneficial (see above). This is easy to do when using a volatile Solvent, but this does not mean that higher-boiling solvents such as e.g. DMF, NMP, DMA or DMSO are not suitable.
  • low-volatile solvents are preferred. These evaporate more slowly and therefore lead to longer reaction times and thus better yields.
  • solvents are suitable here which are suitable for minimizing the aggregation of immobilized substances on the surface, since increased aggregation frequently leads to reduced accessibility of reactive groups and thus to lower reaction yields. This effect has been widely described in peptide synthesis [C. Hyde et al., Int . J. Pept . Prot . Res . 1994, 43 , 431-440].
  • Solvents which are particularly well suited to reducing the aggregation of growing peptide chains and thus to be used in the process according to the invention are, for example, DMF, NMP, DMSO, TFE with DCM or HFIP with DCM [Albericio et al, Methods Enzymol . 1997, 289 , 104-126].
  • the chemical bond that occurs in the process according to the invention between a first functional immobilized on a surface, in particular planar surface Group and a functional group is formed, the second functional Group is part of a compound to be immobilized, is preferably selected from the group that contains carboxylic acid amide bonds, carboxylic acid ester bonds, Carboxylic acid anhydride bonds, sulfonic acid amide bonds, phosphoric acid ester bonds, Phosphonic acid ester bond, phosphoric acid ester bond and phosphoric acid amide bonds includes.
  • the formation of carboxamide bonds is preferred or so-called peptide bonds.
  • the compounds to be immobilized are within the scope of the present invention in particular those compounds which contain a functional group which are suitable is to form one of the chemical compounds mentioned above.
  • Examples of this are carboxylic acids, carboxylic acid esters, sulfonic acids, phosphoric acid esters, phosphonic acid esters, Phosphoric acid esters, phosphoramidites, primary amines, secondary amines and alcohols.
  • Carboxylic acids, primary amines and secondary amines are preferred, and particularly preferably carboxylic acids.
  • Carboxylic acids as immobilized in the process according to the invention are preferably N-terminally protected natural or non-natural Amino acids, particularly preferably N-terminally protected ⁇ -amino acids and very particularly preferably N-terminally protected ⁇ -L-amino acids.
  • the immobilized functional group is a functional group which is suitable is one of the above to form chemical bonds.
  • Examples include carboxylic acids, carboxylic acid esters, Sulfonic acids, phosphoric acid esters, phosphonic acid esters, phosphorous acid esters, Phosphoramidites, primary amines, secondary amines and alcohols.
  • that is immobilized functional group selected from the group consisting of carboxylic acids primary Includes amines and secondary amines.
  • the immobilized is preferably functional Group a primary amine or a secondary amine.
  • There are primary and secondary amines preferably C-terminally protected natural or unnatural amino acids, particularly preferably C-terminally protected ⁇ -amino acids, preferably C-terminally protected ⁇ -L-amino acids.
  • the surface is contacted with a solution. Different methods for this are explained in FIGS. 7 + 8.
  • a principal way of applying liquid is to contact a capillary.
  • a pipette can be used for fully manual procedures.
  • a dispenser in the broadest sense is preferably used.
  • the Auto-Spot-Robot ASP 222 from Abimed Analysentechnik GmbH, Raiffeisenstrasse 3, D-40764 Langenfeld [R. Frank et al. in Combinatorial Peptide and Nonpeptide Libraries, Ed. G. Jung, VCH, Weinheim, 1996, pp. 363-386] can be used.
  • capillaries can also be used to pump liquids without mechanical support, whereby capillary forces determine the suction and delivery of liquid.
  • Suitable capillaries can be those as shown in FIGS. 7B + C.
  • More specific embodiments for contacting a surface with a liquid by contacting with a capillary are, for example, the ⁇ FN process (here a network of many microcapillaries is used) and the ⁇ WP process (Micro Wet Printing from Clondiag [E. Erdmanntraut et al ., Proceedings of ⁇ TAS'98 1998, 217-221, Kluwer Scientific Publishing, Utrecht], in which the use of masks with holes filled with liquid is provided ( ⁇ WP method), both of which are also used as methods
  • ⁇ FN process here a network of many microcapillaries is used
  • ⁇ WP process Micro Wet Printing from Clondiag [E. Erdmanntraut et al ., Proceedings of ⁇ TAS'98 1998, 217-221, Kluwer Scientific Publishing, Utrecht]
  • ⁇ WP method Micro Wet Printing from Clondiag [E. Erdmanntraut et al ., Proceedings of ⁇ TAS'98 1998, 217-221, Kluwer Scientific Publishing
  • Spraying liquids is a possible procedure.
  • Process for spraying on Liquids on surfaces, as they are in the process of the invention can be used are known to those skilled in the art.
  • tubing or needles can be used in combination with a pump or syringe to generate drops.
  • a method that is particularly simple in terms of apparatus is the use of a spray bottle [VWR International, Laboratory Catalog, 2002, p. 215.32].
  • the prototype of a piezoelectric device is a glass capillary surrounded by a cylindrical piezoelectric element [TW Shield, IBM J. Res. Dev. 1987, 31 , 96-100].
  • a short electrical pulse typically 100 V, 5 ⁇ s
  • the piezoelectric element leads to a compression thereof, which in turn generates shock pulses in the liquid chamber.
  • drops are thrown out of the device, as is shown, for example, in FIGS. 8A and 8B.
  • Piezoelectric devices require a relatively large amount of liquid used (typically 1-4 ⁇ l); for this, drops with a very defined size can be generated.
  • a special case of spraying on solutions is the so-called top spot printing
  • the company GeneScanEurope GmbH as shown for example in FIG. 8C.
  • Generated here a pneumatic pulse on a special microtiter plate up to 96 different ones Solution drops in parallel within one second.
  • the main advantage of this procedure is the extremely high speed at which the drops are applied to the surface can be.
  • the method according to the invention applies same solution, e.g. a coupling reagent or a base, with all growing ones Bringing peptide chains into contact.
  • a coupling reagent or a base e.g. a coupling reagent or a base
  • An example is the parallel amino acid coupling by the simultaneous contacting of all membrane-bound amines with a solution of the Coupling reagent HBTU (e.g. 0.3 mol / l dissolved in acetonitrile), drying, and then applying a solution of an amino acid as that to be immobilized Compound and a base (e.g. Fmoc-Gly-OH and DIPEA, 0.6 M and 1.2 M in NMP).
  • the parallel application of the HBTU solution on e.g. 2000 spots e.g. with a suitable one Spray bottle within a few seconds.
  • stamping liquids is a possible procedure.
  • An example of possible One embodiment is contact tip deposition printing (Fig. 7-A). With this procedure immerses the tip of a solid pin in a solution, creating a defined amount of solution is adsorbed on the pin. By bringing the pin tip into contact with the surface the liquid is transferred to the surface. This allows spot sizes manufacture down to 50 ⁇ m.
  • Fig. 7-A contact tip deposition printing
  • FIG. 7-D Another possible embodiment is "pin and ring printing" (Fig. 7-D).
  • both terms are used interchangeably herein used, which is used in the context of the inventive method a mechanically stable, compared to the reagents and solvents used chemically inert flat material used.
  • Flat material is understood to mean a material where one of the three mutually perpendicular spatial dimensions is significantly smaller than the other two dimensions is.
  • This flat material or carrier from it is / are in Within the scope of the method according to the invention with any spatially limited form as Segments used, for example in the form of leaves or strips.
  • the flat material can be porous or non-porous and applied on a support as desired or be self-supporting. Can be used as flat material e.g. Foils, plates, membranes or Papers.
  • the flat material has chemically reactive groups (Functions), which are preferably part of an immobilized connection to which suitable connections can be covalently linked.
  • This is advantageous Carrier material or the surface, which is used for chemical synthesis, too suitable for use in - preferably biological - test reactions.
  • biological Test reactions are used to determine the structure or function of a connection (e.g. a to examine peptide bound to a flat material).
  • An example is the implementation with a fluorescent-labeled antibody that targets specific areas of a peptide binds.
  • the compounds prepared using the methods of the invention can directly in the said without isolation and subsequent immobilization Test reactions are used when linking between to be immobilized Compound and carrier material was chosen so that it was stable during the biological Test reaction is.
  • the flat material is porous, its inner surface also stands for the synthesis is available. Washing operations can then also be carried out by sucking Solutions are done through the material. Non-porous flat material has a lot less surface. Washing operations can be carried out by simply rinsing it off.
  • flat materials made of different materials can be used.
  • the area-specific loading with reactive functions or immobilized compounds can be varied within a wide range (eg 1 pmol to 1 ⁇ mol per cm 2 ).
  • planar surfaces which can be used in the process according to the invention include cellulose [R. Frank et al., Tetrahedron 1988, 44 , 6031-6040] [J. Eichler et al., Collect. Czech. Chem . Commun. 1989 , 54, 1746-1751] [R. Frank, Tetrahedron 1992 , 48 , 9217-9232], cotton [J. Eichler et al., Peptide Res . 1991, 4 , 296-307] [M. Schmidt et al., Bioorg. Med. Chem. Lett . 1993, 3 , 441-446] or polymeric films [RH Berg et al., J. On Chem . Soc .
  • rigid, non-porous materials such as glass, gold-coated surfaces, titanium, aluminum oxide, silicon and modified polymers such as polypropylene, polyethylene, polytetrafluoroethylene or polyvinylidene difluoride surfaces.
  • Fig. 1 shows the principle of solid phase synthesis of peptides.
  • the C-terminal amino acid of the target peptide ie the polypeptide to be produced by carrying out the process according to the invention several times, is first bound to the insoluble support material via the carbonyl group.
  • This can be directly linked to the surface or the amino acid is linked to a linker, which in turn is bound to the surface.
  • Linkers are frequently used in solid phase synthesis with the purpose of ensuring that a compound is stably linked to a solid phase during synthesis and can be split again after the synthesis. Examples of linkers are listed in many reviews [F. Guillier et al., Chem . Rev. 2000, 100 , 2091-2157].
  • Functional groups in the side chain of the amino acid (s) that are already immobilized on the surface must be masked with permanent protective groups that are not impaired by the conditions of peptide synthesis.
  • the temporary protective group for the amino group which protects the already immobilized amino acid during the binding to the solid phase, is removed.
  • An excess of the second amino acid, ie the amino acid to be immobilized or chemically bound to the immobilized amino acid, is introduced, the carboxy group of the amino acid being activated for the formation of the amide bond.
  • excess reagents are removed by washing and the protective group is removed from the N-terminus of the immobilized compound now present as a dipeptide before the third amino acid is added. This process is repeated until the desired peptide sequence is synthesized.
  • the peptide is cleaved from the surface and the side chain protective groups are removed.
  • Log chain protecting groups and the linker are often chosen such that both are split under the same conditions.
  • the peptide can be prepared using the The method according to the invention is also gradually built up starting at the N-terminus become.
  • the activation of solid phase bound carboxylic acids often leads to Racemization, which is why this path is rarely followed, although it is one for some applications is a sensible alternative to synthesis starting from the C-terminus.
  • the method is based on an orthogonal protective group strategy, the base-labile N-Fmoc group being used for the protection of the alpha-amino group and acid-labile side chain protective groups and linkers.
  • the base-labile N-Fmoc group being used for the protection of the alpha-amino group and acid-labile side chain protective groups and linkers.
  • t- butyl and trityl-based side chain protecting groups and alkoxy-benzyl-based linkers are used because they can be removed with TFA.
  • TFA is an excellent solvent for peptides, can be used in standard glass equipment and is volatile enough to be removed in a vacuum.
  • FIG. 2 shows an overview of the Fmoc / t Bu strategy.
  • the C-terminal amino acid is first attached to a TFA-labile linker.
  • the side chains are protected with TFA-unstable protective groups.
  • the temporary N-alpha-Fmoc protective group is then removed with 20% piperidine in DMF.
  • the next amino acid is then usually coupled in DMF or NMP with activated amino acids.
  • the peptide is then cleaved from the linker with 95% TFA and the side chain protective groups are removed at the same time.
  • Fmoc access to SPPS has already been summarized in many reviews [Fields et al., Int. J. Pept. Protein Res. 1990, 35 , 161] [Chan, White, Ed., Fmoc Solid Phase Peptide Synthesis , Oxford University Press, 2000 ].
  • Figure 3 shows the general mechanism of an amide formation reaction.
  • XR XR 'reduces the electron density on the carbonyl carbon atom, so that the nucleophilic attack of the amino component is favored.
  • the nucleophilic amino component XXb attacks the carbonyl carbon atom with the lone pair of electrons and displaces the binding electron pair which is taken up by the carbonyl O atom. This negative charge is electrophilically stabilized within intermediate XXc, which breaks down to form the amide bond.
  • Figure 4 shows the mechanism of peptide bond formation by aminium salts [V. Dourtoglou et al., Synthesis 1984, 572].
  • the more stable of the two intermediates X6 and X7 is the uncharged connection X7.
  • X7 is therefore predominantly in solution and the couplings in the process according to the invention take place starting from this active species [Albericio et al, Methods Enzymol . 1997 , 289 , 104-126].
  • Figure 5 shows the reaction mechanism of peptide bond formation by phosphonium reagents.
  • FIG. 6 shows a schematic illustration of the auto-spot robot ASP 222 , including a detailed illustration of the needle used.
  • FIG. 7 shows the different application methods of or contacting methods for Liquids on surfaces and the setting tools used.
  • Figures 7B and 7C show methods of applying solutions by contacting a capillary;
  • FIG. 7A and 7D show methods of applying solutions by stamping.
  • the capillaries used at the top be both closed and open.
  • Slotted needles, such as in Fig. 7B are also referred to as "split pins".
  • a single needle arrays of several needles can also be used.
  • Fig. 7 A is shown schematically, on a settling tool due to their capillary forces or surface tensions of liquid drops held by contact of the drop with deposited on the surface.
  • 8 shows the various application methods of or contacting methods for Liquids on surfaces and the setting tools used in the event that it is applied by spraying.
  • 8A and 8B embodiments are one Piezoelectric device for spraying solutions shown in which an electrical Pulse to a compression and ultimately the generation of a drop of a liquid leads.
  • 8C describes the top spot printing of the company GeneScanEurope GmbH, which is characterized in that parallel by a pneumatic pulse on a microtiter plate up to 96 liquid drops can be generated.
  • Example 1 Material and methods
  • the reagents used were developed by Advanced ChemTech (Bamberg, Germany), Sigma-Aldrich-Fluka (Deisenhofen, Germany), Bachem (Heidelberg, Germany), J.T. Baker (Phillipsburg, USA), Lancaster (Mühlheim / Main, Germany), Merck Eurolab (Darmstadt, Germany), Neosystem (Strasbourg, France) or Novabiochem (Bad Soden, Germany) purchased and used without further purification. Whatman 50 cellulose membranes (Whatman Maidstone, UK) were used for SPOT synthesis. used, which were cut to the required size.
  • concentrations of the reagents in percent are, unless otherwise specified by volume percent (v / v).
  • the separation was performed on RP-18 column material (Vydac 218 TP5215, 2.1 x 150 mm, 5 ⁇ m, C18, 300 A with guard column (Merk)) at 30 ° C and a flow of 0.3 ml / min using a linear gradient for all chromatograms (5-95% B within 25 min, where A: 0.05% TFA in water and B: 0.05% TFA in CH 3 CN).
  • the dead time between injection and UV detection (HPLC) was 1.65 min, between UV detection and mass detection 0.21 min.
  • the accuracy of the mass spectrometer is approximately ⁇ 0.2 amu.
  • the automatic SPOT synthesis was carried out using the Autospot Robot ASP 222 (Abimed, Langenfeld, Germany) using the Autospot XL Ver control software. 2.02.
  • the necessary control files, in which the location and sequences of the SPOTs were defined, were created with the programs LISA and DIGEN (both: Jerini AG, Berlin, Germany).
  • the washing steps were carried out in stainless steel trays (Merck Eurolab), which were moved on a rocking table (Labortechnik Fröbel, Lindau / Bodensee, Germany). Individual reaction sites (“SPOTs”) were punched out with an office punch and transferred to a microtiter plate or 2.0 ml Eppendorf reaction vessels for further treatment.
  • Thermomixer 5437, centrifuge 5475C and vacuum centrifuge 5301 from Eppendorf were used for the treatment of punched-out SPOTs in Eppendorf vessels.
  • An ultrasonic bath (Sonomatic 300 PC) was used to accelerate the process of dissolving reagents for SPOT synthesis.
  • the degree of derivatization can be reliably measured by measuring the UV absorption of the Dibenzofulven-piperidine adduct C after removal of the Fmoc protective group Determine membrane-bound amino acid, as shown schematically in Fig. 9.
  • a SPOT (0.23 cm 2 ) is punched out and 1.0 ml of piperidine (20% in DMF) is added in a 2 ml Eppendorf reaction vessel.
  • the quantification is carried out as described under Method A.
  • This process provides an Fmoc-amino-derivatized cellulose membrane in which Fmoc-glycine is bound to the surface via a base-labile ester function.
  • the membrane is then washed (DMF, 3 x 10 min; MeOH, 2 x 5 min; DCM, 1 x 2 min; Et 2 O, 1 x 2 min).
  • free amino groups are blocked by acetylation (DMF, 2 x 2 min; DMF / Ac 2 O / DIPEA [70:10:20 (v / v), 2 x 15 min]), and washed again (DMF, 3 x 2 min; MeOH, 2 x 2 min; DCM, 1 x 2 min; Et 2 O, 1 x 2 min) and then air-dried.
  • the degree of derivatization according to AAV 1B is determined on the basis of three SPOTs that were only intended for this purpose.
  • Fig. 10 is the implementation of the manual peptide synthesis according to the Prior art methods using OPfp esters.
  • a 1 x 1 cm grid is drawn in pencil on an amino-derivatized planar surface.
  • the membrane is always treated in a stainless steel instrument tray.
  • the washing steps are carried out on a rocking table while moving the solvent.
  • 2 ⁇ l (on cellulose) or 1 ⁇ l (on PP membrane) of a 0.6 M amino acid pentafluorophenol ester solution ie a previously synthesized active ester
  • NMP 0.6 M amino acid pentafluorophenol ester solution
  • the membrane After washing (DMF, 3 x 2 min; MeOH, 2 x 2 min; DCM, 1 x 2 min; Et 2 O, 1 x 2 min) and drying in air, the membrane is ready for the coupling of the next amino acid, see above that the synthesis cycles can be run through until the desired peptide sequence is reached.
  • the synthesis can be interrupted or stopped after the amino acids have been coupled or acetylation.
  • This regulation describes how to start from a surface to which a number of connections were consecutively immobilized via a linker structure, and thus a synthesis product was obtained, the synthesis product by splitting off from the Linker structure is released.
  • the carrier material here the membrane, on which peptides were synthesized according to AAV 3 or AAV 4
  • TFA [TFA / H 2 O / TIPS [95/3/2 (v / v)] with the addition of Phenol (1 g / 100ml), 2 x 15 min; TFA / CH 2 Cl 2 / H 2 O / TIPS [50/45/3/2 (v / v)] with the addition of phenol (1 g / 100ml), 1 x 2 h].
  • the instrument tray used is not shaken.
  • the membrane is then washed (CH 2 Cl 2 , 2 x 5 min; DMF, 3 x 2 min; MeOH, 2 x 2 min; Et 2 O, 1 x 2 min) and air-dried.
  • the peptides bound to the cellulose via an ester bond are split off by methylamine in a desiccator within 14 h or by treatment with 0.1 M TEA / H 2 O for 30 minutes.
  • spots The areas of the membrane on which a peptide was synthesized (“spots") are punched out and peeled off in a suitable solvent for assays in solution used or analyzed (HPLC, HPLC-MS).
  • the adsorbed synthesis products are removed with acetonitrile (50% in H 2 O with 0.1% TFA, 100 ⁇ l) and analyzed (HPLC, HPLC-MS, 10 ⁇ l injection volume).
  • Example 2 Comparison between preactivations of the compound to be immobilized on the surface of a carrier material and in solution with HBTU (1 eq.) And DIPEA (2 eq.)
  • various peptides were firstly used using the inventive method, d. H. by pre-activating those to be immobilized Connection (i.e. the respective amino acid) on the surface of a carrier material, more precisely a membrane, shown in FIGS. 11A to 11E as a) and through Preactivation of the compound to be immobilized (i.e. the respective Amino acid) in solution, shown in Figures 11A to 11E as b) synthesized.
  • the peptides listed in FIGS. 11A-E were synthesized according to the following protocol:
  • the coupling reagent HBTU is dissolved in a concentration of 0.3 M in acetonitrile. Each 1.2 ⁇ l of this solution is spotted on the reaction surface of a glycine ester-modified cellulose membrane using an automatic spotter (Abimed ASP 222) . After the solvent has evaporated (approx. 20 min), the process is repeated. Again, the acetonitrile is allowed to evaporate and a solution of the compound Fmoc-AS-OH (0.6 M) and the base DIPEA (1.2 M) in NMP (1.2 ⁇ l) is spotted twice at intervals of about 30 min.
  • the entire membrane is washed 5 times with DMF, 3 times with methanol, acetylated according to AAV 3 and the Fmoc protective group is split off according to AAV 3.
  • the entire process is repeated with the required amino acid until the desired peptide is synthesized.
  • the peptide is cleaved from the cellulose (AAV 5) and analyzed by HPLC. The chromatograms obtained are shown in Fig. 11 AE. The arrow shows the desired product.
  • the peptides listed in FIG. 11A-E were synthesized according to the following protocol: A solution of the coupling reagent HBTU (0.6 M) in NMP and a solution of the compound to be coupled Fmoc-AS-OH (0.6 M) in NMP are 1: 1 by volume mixed and with 2 eq. the base DIPEA. After standing for approx. 45 minutes, 1.2 ⁇ l of this solution is spotted onto the reaction surface of a glycine ester-modified cellulose membrane using an automatic spotter ( Abimed ASP 222 ). This process is repeated after approx.
  • Example 3 Comparison between preactivations of the compound to be immobilized on a surface of a carrier material and in solution with HBTU (1 eq.) And DIPEA (2 eq.)
  • various peptides were firstly used using the inventive method, d. H. by pre-activating those to be immobilized Connection (i.e. the respective amino acid) on the surface of a carrier material, more precisely a membrane, shown in Figures 12A to 12C as a), and on the other by pre-activating the compound to be immobilized (i.e. the respective Amino acid) in solution, shown in FIGS. 12A to 12C as b).
  • FIGS. 12A-C The peptides listed in FIGS. 12A-C were synthesized according to the following protocol: A glycine ester-modified cellulose membrane (52 cm 2 ) is treated with 1 ml of a saturated solution of the coupling reagent HBTU in acetonitrile. After evaporation of the solvent (approx. 20 min), 1.2 ⁇ l of a solution of the compound to be coupled Fmoc-AS-OH (0.6 M) and the base DIPEA (1.2 M) in NMP are spotted on using an automatic spotter (Abimed ASP 222) the reaction area of the membrane. After 15 minutes, the same solution is applied again to the reaction surface and the mixture is waited for a further 15 minutes.
  • the entire membrane is washed 5 times with DMF, 3 times with methanol and the Fmoc protective group is removed in accordance with AAV 3.
  • the entire process is repeated with the required amino acid until the desired peptide is synthesized.
  • the peptide is cleaved from the cellulose (AAV 5) using TEA / water and analyzed by HPLC-MS. The chromatograms obtained are shown in Figures 12A-C. The arrow shows the desired product.
  • the peptides listed in FIG. 12A-C were synthesized according to the following protocol: A solution of the coupling reagent HBTU (0.6 M) in NMP and a solution of the compound to be coupled Fmoc-AS-OH (0.6 M) in NMP are 1: 1 by volume mixed and with 2 eq. The base DIPEA offset. After shaking for about 5 minutes, 1.2 ⁇ l of this solution is spotted onto the reaction surface of a glycine ester-modified cellulose membrane using an automatic spotter ( Abimed ASP 222 ). This process is repeated after approx.
  • Example 4 Influence of the preactivation time on the coupling time for preactivations on the membrane and in solution with HBTU (1 eq.) And DIPEA (2 eq.)
  • the entire membrane is washed 5 times with DMF, 3 times with methanol and the Fmoc protective group is removed in accordance with AAV 3.
  • the peptide is cleaved from the cellulose (AAV 5) using TEA / water and analyzed by HPLC-MS. The chromatograms obtained give the proportion of the desired product RRFG.
  • the educt RFG was identified as a by-product.
  • the pretreatment of the membrane with HBTU according to the invention has clear advantages compared to the conventional preactivation method in solution. So you get already with a short preactivation time (5 min) with method b) approx. 5% more desired product than with method a).
  • Activation times approximately 4-7.5 hours. These activation times occur when very many peptides (e.g. 600) are synthesized simultaneously on a planar membrane.
  • help Method a) used up to now is only about half after 4 hours, after 8 Hours even only about a tenth of what can be achieved with a short pre-activation time Product yield.
  • Process b) leads to yields which are almost independent of Are pre-activation time. Even after a very long pre-activation time of 7.5 hours you get the desired product RRFG in a good yield of 45%, which is approx corresponds to ten times the amount of the yield according to method a).
  • Example 5 Comparison of the synthesis yield in the case of preactivations on a surface of a support material with HBTU (1 eq.) And DIPEA (2 eq.) And pre-synthesized OPfp esters
  • the peptides listed in Table 1 were synthesized according to the following protocol:
  • the coupling reagent HBTU and the respective compound Fmoc-AS-OH to be coupled are dissolved in a concentration of 0.5 M in DMF.
  • Each 1.2 ⁇ l of this solution is spotted on the reaction surface of a glycine ester-modified cellulose membrane using an automatic spotter ( Abimed ASP 222 ).
  • an automatic spotter Abimed ASP 222
  • a solution of the base DIPEA (0.9 M) in NMP 1.2 ⁇ l
  • the membrane is left for 30 minutes, blow-dried for 1-2 minutes and the entire process is repeated.
  • the peptides listed in Table 1 were synthesized according to the following protocol: A solution of commercially available Fmoc-AS-OPfp (0.6 M) in NMP (1.2 ⁇ l) is spotted on the reaction surface of a glycine ester using an automatic spotter ( Abimed ASP 222 ). modified cellulose membrane. This process is repeated after approx. 45 min, left for a further 45 min, the entire membrane is washed 5 times with DMF, 3 times with methanol, acetylated according to AAV 3 and the Fmoc protective group is split off according to AAV 3. The entire process is repeated with the required amino acid until the desired peptide is synthesized.
  • Example 6 Comparison of the purity of the synthesis product in the case of preactivations on a surface of a support material with HBTU (1 eq.) And DIPEA (3 eq.) And preactivation of the compound to be immobilized in solution
  • the peptides listed in Table 2 were synthesized according to the following protocol:
  • the coupling reagent HBTU and the compound to be coupled Fmoc-AS-OH are dissolved in a concentration of 0.5 M each in DMF.
  • An automated spotter Abimed ASP 222 ) spotted 1.2 ⁇ l of this solution onto the reaction surface of a glycine ester-modified cellulose membrane on which the TF-G peptide sequence was synthesized according to AAV-5. After waiting 10 minutes and blow drying for one minute with a commercially available hair dryer, a solution of the base DIPEA (1.35 M) in NMP (1.2 ⁇ l) is spotted on it. Allow the membrane to lie for 30 minutes, blow dry for 1-2 minutes and repeat the entire process.
  • the entire membrane is washed 5 times with DMF, 3 times with methanol, acetylated according to AAV 3 and the Fmoc protective group is split off according to AAV 3.
  • the entire process is repeated with the required amino acid until the desired peptide is synthesized.
  • the peptide is split off from the cellulose (AAV 5) and analyzed by HPLC. The purities of the peptides obtained are shown in Table 2.
  • the peptides listed in Table 2 were synthesized according to the following protocol: A solution of HBTU (0.6 M) in NMP and a solution of Fmoc-AS-OH (0.6 M) in NMP are mixed in a volume ratio of 1: 1 and mixed with 3 eq. DIPEA offset. After standing for approx. 45 minutes, 1.2 ⁇ l of this solution is spotted onto the reaction surface of a glycine ester-modified cellulose membrane using an automatic spotter ( Abimed ASP 222 ). This process is repeated after approx.
  • the entire membrane is washed 5 times with DMF, 3 times with methanol, acetylated according to AAV 3 and the Fmoc protective group is split off according to AAV 3. The entire process is repeated with the required amino acid until the desired peptide is synthesized. After renewed acetylation, the peptide is split off from the cellulose (AAV 5) and analyzed by HPLC. The purities of the peptides obtained are shown in Table 2.
  • Example 7 Influence of various solvents on the coupling efficiency when the compound to be immobilized is preactivated on the surface
  • the coupling reagent HBTU is in the maximum possible concentration in each Solvent dissolved. Each 1.2 ⁇ l of this solution is spotted on the reaction surface of one Glycine ester-modified cellulose membrane. After evaporation of the solvent (approx. 20 min) the process is repeated. Again, the acetonitrile is allowed to evaporate and then mocks a solution of the or immobilized twice at intervals of approx. 30 min to the coupling compound Fmoc-Gly-OH (0.6 M) and the base DIPEA (1.2 M) in NMP (1.2 ⁇ l).
  • the entire membrane is washed 5 times with DMF, 3x with methanol, 2x with dichloromethane and determines the yield of the coupling Amino acid by photometric quantification of the membrane-bound Fmoc.
  • Example 8 NMR investigations for the preactivation of Fmoc-Ala-OH with HBTU in the presence or absence of base
  • the carboxylic acid Fmoc-Ala-OH and the coupling reagent HBTU are each 0.45 M dissolved in DMF-d7 (0.8 ml) and the solution is transferred to a sealed NMR tube. After 15 min and standing for one day at room temperature, a 13 C-NMR spectrum is recorded, which is shown in FIGS. 14A-B. The base DIPEA (0.90 M) is then added and a 13 C-NMR spectrum is measured after 10 min and one day (FIG. 14 C-D).
  • Example 9 Influence of different storage conditions on the activity of solutions from different amino acids and the coupling reagent HBTU
  • the Fmoc-AS-OH is coupled to the free amino group of the H-Gly-OEt instead. If there is no under the storage conditions HBTU has decomposed, the coupling runs completely, and only the HPLC spectrum is Compound Fmoc-AS-Gly-OEt detectable. But if part of the HBTU becomes inactive Compounds reacted, the HPLC spectrum shows next to the desired Coupling product Fmoc-AS-Gly-OEt also free amino acid Fmoc-AS-OH.
  • the proportion of the desired coupling product in the peak areas in the HPLC is as Activity or residual activity of the respective solution of Fmoc-AS-OH, HBTU (and. Possibly. DIPEA).
  • Fig. 15 shows the activity of solutions different amino acids and the coupling reagent HPTU in different Storage conditions shows.
  • 15A shows Fmoc-AS-OH, HBTU (each 0.45 M in DMF) and Fig. 15B Fmoc-AS-OH, HBTU (0.45 M each in DMF), DIPEA (1.2 M).

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006225270A (ja) * 2005-02-15 2006-08-31 Yoshihiro Futamura 性ホルモン修飾作用を有するトリペプチド又はそれを含有する抽出物、エイコサペンタエン酸を結合してなるトリペプチド、それからなる食品製剤、化粧品製剤、子宮内膜症治療剤
US10087221B2 (en) 2013-03-21 2018-10-02 Sanofi-Aventis Deutschland Gmbh Synthesis of hydantoin containing peptide products
US10450343B2 (en) 2013-03-21 2019-10-22 Sanofi-Aventis Deutschland Gmbh Synthesis of cyclic imide containing peptide products
CN111157739A (zh) * 2019-12-28 2020-05-15 王贤俊 Ⅳ型胶原蛋白抗体胶乳颗粒及其制备方法及其专用组合型偶联剂
WO2020123714A1 (fr) * 2018-12-12 2020-06-18 Purilogics, LLC Membrane d'affinité et son procédé de préparation
CN113668069A (zh) * 2020-05-13 2021-11-19 洛阳中科生物芯片技术有限公司 一种蛋白质芯片板的制备方法
CN115656494A (zh) * 2022-10-17 2023-01-31 济南诺磐生物科技有限公司 一种蓝色微球偶联抗体的方法及应用

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EP0134041A1 (fr) * 1983-08-25 1985-03-13 Akademie der Wissenschaften der DDR N-Chlorocarbonyloxy norbornène-5 dicarboximide-2,3, procédé pour sa préparation et son utilisation
WO1992004366A1 (fr) * 1990-08-31 1992-03-19 GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) Procede et dispositif de synthese rapide de peptides ou d'oligonucleotides libres ou immobilises sur des substrats, materiau plat ainsi obtenu et son utilisation
WO1994015948A1 (fr) * 1993-01-11 1994-07-21 Applied Biosystems, Inc. Support et procede pour immobiliser des polypeptides
US5874569A (en) * 1992-08-10 1999-02-23 Mouritsen & Elsner A/S Method of preparing tresyl-activated dextran, article having tresyl-activated dextran fixed covalently to its surface, and immobilization of chemical compounds thereto
US6277957B1 (en) * 2000-03-23 2001-08-21 Derek Hudson Method for production of acylthio derivatives
WO2002053606A1 (fr) * 2001-01-04 2002-07-11 Council Of Scientific And Industrial Research Nouveau support solide contenant un lieur pour la synthese organique de peptides et de petites molecules

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EP0134041A1 (fr) * 1983-08-25 1985-03-13 Akademie der Wissenschaften der DDR N-Chlorocarbonyloxy norbornène-5 dicarboximide-2,3, procédé pour sa préparation et son utilisation
WO1992004366A1 (fr) * 1990-08-31 1992-03-19 GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) Procede et dispositif de synthese rapide de peptides ou d'oligonucleotides libres ou immobilises sur des substrats, materiau plat ainsi obtenu et son utilisation
US5874569A (en) * 1992-08-10 1999-02-23 Mouritsen & Elsner A/S Method of preparing tresyl-activated dextran, article having tresyl-activated dextran fixed covalently to its surface, and immobilization of chemical compounds thereto
WO1994015948A1 (fr) * 1993-01-11 1994-07-21 Applied Biosystems, Inc. Support et procede pour immobiliser des polypeptides
US6277957B1 (en) * 2000-03-23 2001-08-21 Derek Hudson Method for production of acylthio derivatives
WO2002053606A1 (fr) * 2001-01-04 2002-07-11 Council Of Scientific And Industrial Research Nouveau support solide contenant un lieur pour la synthese organique de peptides et de petites molecules

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006225270A (ja) * 2005-02-15 2006-08-31 Yoshihiro Futamura 性ホルモン修飾作用を有するトリペプチド又はそれを含有する抽出物、エイコサペンタエン酸を結合してなるトリペプチド、それからなる食品製剤、化粧品製剤、子宮内膜症治療剤
US10087221B2 (en) 2013-03-21 2018-10-02 Sanofi-Aventis Deutschland Gmbh Synthesis of hydantoin containing peptide products
US10450343B2 (en) 2013-03-21 2019-10-22 Sanofi-Aventis Deutschland Gmbh Synthesis of cyclic imide containing peptide products
WO2020123714A1 (fr) * 2018-12-12 2020-06-18 Purilogics, LLC Membrane d'affinité et son procédé de préparation
US11918957B2 (en) * 2018-12-12 2024-03-05 Donaldson Company, Inc. Affinity membrane and method of preparation
CN111157739A (zh) * 2019-12-28 2020-05-15 王贤俊 Ⅳ型胶原蛋白抗体胶乳颗粒及其制备方法及其专用组合型偶联剂
CN113668069A (zh) * 2020-05-13 2021-11-19 洛阳中科生物芯片技术有限公司 一种蛋白质芯片板的制备方法
CN113668069B (zh) * 2020-05-13 2023-12-08 洛阳中科生物芯片技术有限公司 一种蛋白质芯片板的制备方法
CN115656494A (zh) * 2022-10-17 2023-01-31 济南诺磐生物科技有限公司 一种蓝色微球偶联抗体的方法及应用

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